THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 104 MOD: 1st Issue Page: 1 of 8

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1 THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 104 MOD: 1st Issue Page: 1 of 8 Procedure Type: General Laboratory Procedure 1. Introduction Gene expression and protein production using retrovirus requires the production, handling, and storage of infectious retrovirus. Recombinant retroviruses can be hazardous depending on the retroviral inserts and the tropism of the virus. It is imperative to fully understand the potential hazards of and necessary precautions for the laboratory use of retroviruses. Tropism refers to the host range of a particular virus, and is dependant on the interaction of specific cell surface molecules with a protein on the surface of the viral particle. The specificity of the interaction of the virus envelope with host receptors prevents superinfection of host cells with wild-type virus, as the Env protein being produced in the host cell will interact with the receptor in the endoplasmic reticulum and prevent processing to the cell membrane. Tropism can be categorized by the range of host cells that can be infected: Ecotropic virus replicate only in cells from the natural host or closely related species. For example, packaging cells expressing the Moloney murine leukemia virus (MMLV) ecotropic envelope would produce virus that would allow transduction of rat or mouse cells only. The MMLV ecotropic envelope recognizes a novel receptor protein known as mcat-1. It is a cationic amino acid transporter in mammalian cells which is primarily expressed in mouse and rat cells. Amphotropic virus replicate in cells of BOTH the natural host and foreign origin (ie, they are potentially infectious to humans), as the amphotropic virus receptor is expressed in multiple species, including the host species. Xenotropic virus replicate efficiently ONLY in cells of foreign origin, not the host, as the xenotropic virus receptor is expressed in multiple species, but not the host species. Pantropic virus replicate efficiently in ANY cells. These viruses are able to enter the cell independent of cell surface receptors or are able to attach to more than one receptor. The vectors described here are MMLV-based vectors and do not contain Env viral components, and thus cannot naturally infect human cells; however, viruses packaged from the MMLV-based vectors described here are capable of infecting human cells if packaged in a cell line with the proper tropism. To date, no documented clinical manifestations of disease have been noted in humans exposed to MMLV vectors. In vivo infection in humans appears to require direct injection with amphotropic, pantropic or pseudotyped virus. WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Ellen Byrnes Lynn Herd Phillip Dickson DATE 10 th February nd March th January 2007 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 8 2. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Use of an Ecotropic Retroviral Expression System for the Infection of Target Cells HAZARDS 1. Viruses packaged from MMLV-based vectors described here are potentially capable of infecting human cells - BIOHAZARD 2. Working with genetically modified cell lines RISK ASSESSMENT 1. The risk of exposure to viruses capable of infecting humans is minimized by the conditions under which these viruses are produced and the cell-lines into which the viruses are introduced. When following this protocol, any viruses produced should be unable to infect human tissues. The risk is considered to be low providing all of the Risk Controls are followed. 2. The risk of working with genetically modified cell-lines is low providing all of the Risk Controls are followed. 3. The risks involved with this work must be re-assessed each time a new cell-line is introduced to ensure that the target cell-line lacks the viral structural and polymerase/integrase genes necessary for viral replication. RISK CONTROL 1. Approval must be obtained from the University of Newcastle Institutional Biosafety Committee and the OGTR before any work involving the use of this procedure may proceed. Changes to the procedure involving the use of different cell-lines or vectors must be approved by the IBC before any further work proceeds. 2. Always practice safe laboratory procedures 3. All work must be performed in a Physical Containment Level 2 Tissue Culture Facility 4. Use a Class II Biosafety cabinet with HEPA filter 5. Limit access to the areas in which this work is to be performed. All work should take place in the Retroviral Laboratory LS Use appropriate PPE - double gloves plus a lab coat and safety glasses 7. Use ethanol, detergents, or bleach to inactivate retroviral vectors eg. Sodium hypochlorite with final volume 1-3% active chlorine, detergents at a specific concentration which you can document as suitable to inactivate the vectors, 70% ethanol.

3 Page: 3 of 8 8. Autoclave all waste not treated with 1-3% sodium hypochlorite for decontamination prior to disposal. 9. Post biohazard warning signs on doors to facility, and on all equipment which will be used during this work. 10. Minimize production of aerosols. Packaging cells and infected cells should not be used outside a biohazard hood until proven that no infectious virus particles are being produced. When pipetting virus-containing supernatants and transferring plates or flasks to and from the biohazard hood, the production of aerosols should be avoided. 11. Take precautions with sharps, in particular needles and dispose of them promptly into a sharps container for autoclaving. 12. Refer to GEP005 Biohazard Hood for any procedures involving the use of the Biohazard hood. 13. All persons using the Autoclave must have received training in its operation and have read the relevant equipment procedure. 14. Training must be provided by personnel experienced in this procedure or in similar retroviral work. 15. All personnel working with these materials must read and sign the approval from the OGTR / IBC. 16. Training must be undertaken in General Laboratory Safety, Microbiological Safety and Molecular Biology Safety.

4 Page: 4 of 8 I have read and understood the Risk Assessment for GLP Ecotropic Retroviral Infection. I agree to abide by the Risk Controls outlined in this Assessment. Name Signature Date

5 Page: 5 of 8 3. Purpose: 3.1. Infection of hard-to-transfect rodent cell lines 3.2. Production of transgenic target cells stably expressing an integrated gene of interest 4. Equipment: 4.1. Class II Biosafety Cabinet with a HEPA microfilter 4.2. Laboratory gown 4.3. Safety glasses 4.4. Disposable gloves latex, nitrile or vinyl 4.5. Tissue culture incubator 37 o C, 5% CO Eppendorf centrifuge 4.7. Autoclave 5. Materials: 5.1. Low protein-binding 0.45µ filter - Millipore, Billerica, MA, USA 5.2. autoclave bags double bagging mm tissue-culture plates mm 2 filter-capped tissue culture flasks ml microcentrifuge tubes ml sterile yellow-capped tubes 6. Reagents: 6.1. Lipofectamine Invitrogen Opti-MEM media - Invitrogen Growth media - usually 4 ml DMEM / 2% FCS without antibiotics 6.4. DMEM/10% FCS containing 8 µg/ml polybrene (Chemicon, Temecula, CA, USA) Geneticin (G418) Invitrogen PBS 6.7. Crystal violet solution - Toxic 7. Cell-lines / Vectors: T or BOSC23 packaging cells 7.2. MMLV-based retroviral plasmid (eg prufneo; MSCV-IRES-GFP) containing the gene of interest 7.3. pgp (gag-pol-expressing vector) 7.4. peco-r (ecotropic env-expressing vector) 7.5. NIH-3T3 mouse fibroblast cells 7.6. FDC-P1 cells line (free of the viral components required to produce an infectious viral particle through a recombination event).

6 Page: 6 of 8 8. Set Up: 8.1. Ensure that you have read and understood the Safety Precautions (Section 9 of this SOP) prior to commencing this procedure. 9. Safety Precautions: 9.1. Good laboratory techniques are to be used at all times Autoclave for decontamination 9.3. Use un-recirculated exhaust air. All work to be carried out in isolated PC3 laboratory equipped with separate air supply Stock chemical disinfectants for spills (see 9.6) 9.5. Use a separate tissue culture incubator which is reserved specifically for this work only Susceptible to 1% sodium hypochlorite, 1% SDS, 70% ethanol. Recommend fresh solution of 10% bleach for 15 minutes Viral particles are extremely labile and do not survive on environmental surfaces First Aid/Treatment: For splashes to the eye of material containing virus, rinse eye at eyewash for 15 minutes. In the case of accidental injection of material containing virus, wash area well with soap and water then contact office of Occupational Health and request serum sample Spills: Allow aerosols to settle for 15 minutes; wear protective clothing and gently cover the spill with adsorbent paper towel and apply freshly prepared 10% sodium hypochlorite starting at the perimeter and working towards the centre; allow at least 15 minutes contact time before clean up Always dispose of lab gown when finished work - do not walk around laboratory in gown used for retroviral work Any material that has been in contact with retrovirals must be treated as infectious. 10. Method: This protocol consistently results in the production of viral titers >10 7 colony forming units (cfu)/ml when transducing NIH3T3 cells with a MoMuLV-derived vector Retroviral Production Split 293T cells at or BOSC23 cells at x 10 6 per 60-mm tissue culture plate in growth medium (usually 4 ml DMEM/2% FCS without antibiotics) 24 hours before the transfection and incubate at 37 C until needed. This should achieve a cell density of between 60-80% the following day.

7 Page: 7 of Pipette the following into a clean 1.5-ml microcentrifuge tube containing 500µl Opti-MEM media (Invitrogen); prepare one tube for each transfection to be carried out. 3.5 µg an MMLV-based retroviral plasmid containing the gene of interest 2-3 µg pgp vector (gag-pol-expressing vector) 2-3 µg of the env-expressing vectors, peco-r Plasmid DNA preparations of high purity should be used for transfections. NOTE: BOSC23 cells are permanently transfected with gag-pol and ecotropic env, so the pgp and peco-r vectors are not essential, but may improve transfection efficiency. To transfect cells without these helper vectors, use 4-5µg of MMLV-based retroviral plasmid DNA µl of Lipofectamine 2000 (Invitrogen) is diluted in 500µl Opti-MEM I Reduced Serum media and incubated for 5 minutes at room temperature The diluted Lipofectamine 2000 is added drop-wise to the DNA mixture and incubated for 20 minutes at room temperature. This complex was then added drop-wise to the retroviral packaging cells and incubated for 5 hours, after which time the media was replaced with fresh DMEM/10% FCS hours post infection, the viral-containing supernatant was carefully collected with a disposable Pasteur pipette and filtered through a 0.45 µm filter to remove particulate matter (Millipore), and used for target cell infection and retroviral titre determination. Retroviral Titre Determination The retroviral titre measures the number of infectious viral particles per ml of supernatant To determine the retroviral titre produced from BOSC23 and 293T packaging cells, viral supernatants are thawed rapidly at 37 o C and diluted 1:100 and 1:1000 in 2 ml DMEM/10% FCS containing 8 µg/ml polybrene (Chemicon) in a sterile 5ml yellow-capped tube The diluted viral medium is overlaid onto NIH-3T3 mouse fibroblast cells seeded the day before at 1 x 10 5 cells/well of a 6-well plate. The cells are incubated for 24 hours before the viral supernatant is replaced with fresh culture medium and incubated for a further 24 hours Cells are harvested and sub-cultured 1:50 and 1:100 in DMEM/10%FCS containing 400 µg/ml of G418. Culture media is replaced every 3-4 days for 10 days to allow antibiotic-resistant colonies to form Medium is removed and cells washed twice in PBS, followed by incubation with crystal violet solution for 10 minutes to stain the colonies. Cells are washed twice in Milli-Q water, and the number of colonies counted. Viral titre (colony forming units (CFU)/ml) is calculated using the following formula: CFU/ml = number of G418 resistant (G418 R ) colonies x dilution of viral supernatant x dilution of cells

8 Page: 8 of 8 Infection of Cells with Viral Supernatant FDC-P1 cells are centrifuged at 220 x g for 5 minutes in an Eppendorf 5810 centrifuge and resuspended at 2 x 10 5 cells/ml in a mixture containing 3ml DMEM/10%FCS and 1 ml of rapidly thawed viral supernatant supplemented with 8 µg/ml polybrene and placed into a 45 mm 2 tissue culture flask Cells are incubated for 24 hours and the viral supernatant replaced with fresh culture medium. Cells are incubated for a further 24 hours, harvested and resuspended in selection medium containing 1 mg/ml G418. Media is replaced every 3-4 days until un-infected control cells had died. Successfully infected cells are expanded and cryopreserved. 11. Maintenance: Shutdown: All solid waste to be double-bagged and placed into a secondary container for transport and autoclaved prior to disposal. Liquid waste to be incubated in 10% bleach solution for at least 15 min prior to disposal in contaminated waste bins. 13. References: pv Pack Vectors Instruction Manual - Stratagene 14. Change History: Issue Number: 1st Issue Date Issued: 30 th January Issue Number: Date Issued: Reason for Change: